This proposal describes experiments designed to determine how 100 or more non-tubulin proteins of the flagellar axoneme (NTAP) are regulated during the differentiation of Naegleria gruberi amebae into swimming flagellates. This question will be examined directly at the level of protein synthesis by determining the degree of chase of 35S from NTAP when cells grown on 35S labeled bacteria are allowed to differentiate in a high concentration of methionine. Quantitative and qualitiative changes in the mRNA population will be examined by hydridization of cDNA copies to poly(A) plus RNA prepared from various stages of the differentiation and various subcellular fractions. The amount and sequence complexity of poly(A) minus mRNA will also be examined by hybridization to single copy DNA. The arrangement of genes coding for differentiation specific functions will be examined by cloning cDNA copies of their mRNAs. These cloned sequences will be used to probe a library of cloned Naegleria DNA in order to study the distribution of these genes. Cloned cDNA sequences will also be used to study the kinetics and distribution of the mRNA sequences during the differentiation.